Compositions and methods for detecting lung cancer

ABSTRACT

The present invention relates to compositions and methods for the in vitro diagnosis of lung cancer, wherein said compositions comprise an antibody binding to progastrin and said methods comprise the use of an antibody binding to progastrin.

INTRODUCTION

The present invention relates to the in vitro diagnosis of cancer, moreparticularly it relates to methods for the in vitro diagnosis of lungcancer. Compositions according to the invention comprise aprogastrin-binding molecule, in particularly an anti-hPG antibody,whereas methods according to the invention comprise the use of aprogastrin-binding molecule, and particularly to an anti-hPG antibody.

Lung cancer remains the most lethal malignancy in the world. Despiteimprovements in surgical treatment, systemic therapy, and radiotherapy,the 5-year survival rate for all patients diagnosed with lung cancerremains between 15 and 20%.

Lung cancer comprises two main types of tumors, namely small cell lungcancer (SCLC) and non-small cell lung cancer (NSCLC). SCLC represents15-18% of all lung cancers, while NSCLC make up about 80% to 85% of lungcancers. Other types of lung cancer such as adenoid cystic carcinomas,lymphomas, and sarcomas, as well as benign lung tumors such ashamartomas are rare.

Small cell and non-small cell lung cancers are treated differently. Inparticular, SCLC is more responsive to chemotherapy and radiationtherapy than other cell types of lung cancer. However, a cure isdifficult to achieve because SCLC has a greater tendency to be widelydisseminated by the time of diagnosis. To date, there are no molecularbiomarkers that have been translated to widespread clinical practice oflung cancer. Treatments depend on the development of the cancer, andusually include surgery, for small-localized tumors, or chemotherapy,possibly in combination with radiation therapy.

Therefore, there is still a need for methods allowing a quick, reliableand cost-effective diagnosis of lung cancer.

This is the object of the present invention.

DESCRIPTION

The present invention now provides methods for the in vitro diagnosis oflung cancer, wherein said method comprises the detection progastrin in abiological sample from a subject. Preferably, the amount of progastrinin said sample is determined, thus allowing quantification ofprogastrin.

Human pre-progastrin, a 101 amino acids peptide (Amino acid sequencereference: AAB19304.1), is the primary translation product of thegastrin gene. Progastrin is formed by cleavage of the first 21 aminoacids (the signal peptide) from preprogastrin. The 80 amino-acid chainof progastrin is further processed by cleavage and modifying enzymes toseveral biologically active gastrin hormone forms: gastrin 34 (G34) andglycine-extended gastrin 34 (G34-Gly), comprising amino acids 38-71 ofprogastrin, gastrin 17 (G17) and glycine-extended gastrin 17 (G17-Gly),comprising amino acids 55 to 71 of progastrin.

Anti-human progastrin (anti-hPG) monoclonal antibodies and their use fordiagnosis or therapy have been described in the following documents: WO2011/083 088 for colorectal cancer, WO 2011/083 090 for breast cancer,WO 2011/083 091 for pancreatic cancer, WO 2011/116 954 for colorectaland gastrointestinal cancer, and WO 2012/013 609 and WO 2011/083089 forliver pathologies.

The present invention will become more fully understood from thedetailed description given herein and from the accompanying drawings,which are given by way of illustration only and do not limit theintended scope of the invention.

In a first aspect, the present invention relates to a method for the invitro evaluation of a risk of the presence of lung cancer, wherein saidmethod comprises a step of detecting progastrin in a biological samplefrom a subject. The presence of progastrin in the sample indicates thatthere is a risk of the presence of lung cancer.

Thus, in a first embodiment, the invention relates to an in vitro methodfor evaluating the risk of the presence of lung cancer in a subject,said method comprising the steps of:

a) contacting a biological sample from said subject with at least oneprogastrin-binding molecule, and

b) detecting the binding of said progastrin-binding molecule toprogastrin in said sample, wherein said binding indicates a risk of thepresence of lung cancer.

The binding of progastrin-binding molecule may be detected by variousassays available to the skilled artisan. Although any suitable means forcarrying out the assays are included within the invention, it can bementioned in particular FACS, ELISA, RIA, western-blot and IHC.

In a preferred embodiment, the method according to the invention for thein vitro evaluation of a risk of the presence of lung cancer in asubject, comprises the steps of:

a) contacting said biological sample from said subject with at least oneprogastrin-binding molecule,

b) determining the concentration of progastrin in said biologicalsample, wherein a concentration of progastrin of at least 10 pM in saidbiological sample is indicative of a risk of the presence of lungcancer.

Once the concentration of progastrin present in the sample isdetermined, the result can be compared with those of control sample(s),which is (are) obtained in a manner similar to the test samples but fromindividual(s)s known not to suffer from a lung cancer. If theconcentration of progastrin is significantly more elevated in the testsample, it may be concluded that there is an increased likelihood thatthe subject from whom it was derived has a lung cancer.

Thus, in a more preferred embodiment, the method of the inventioncomprises the further steps of:

c) determining a reference concentration of progastrin in a referencesample,

d) comparing the concentration of progastrin in said biological samplewith said reference concentration of progastrin,

e) evaluating, from the comparison of step d), the risk of the presenceof lung cancer.

According to another aspect, the invention relates to an in vitro methodfor diagnosing lung cancer in a subject, said method comprising thesteps of:

a) contacting a biological sample from said subject with at least oneprogastrin-binding molecule, and

b) detecting the binding of said progastrin-binding molecule toprogastrin in said sample, wherein said binding indicated the presenceof lung cancer in said subject.

In a preferred embodiment, the present invention relates to a method forthe in vitro diagnosis of lung cancer in a subject, comprising the stepsof:

a) contacting said biological sample from said subject with at least oneprogastrin-binding molecule,

b) determining concentration of progastrin in said biological sample,wherein a concentration of progastrin of at least 10 pM in saidbiological sample is indicative of the presence of lung cancer in saidsubject.

In a more particular embodiment of a method according to the invention,a concentration of progastrin of at least 10 pM, at least 20 pM, atleast 30 pM, in said biological sample is indicative of the presence oflung cancer in said subject.

In a more preferred embodiment, the method of the invention comprisesthe further steps of:

a) determining a reference concentration of progastrin in a referencesample,

b) comparing the concentration of progastrin in said biological samplewith said reference level or concentration of progastrin,

c) diagnosing, from the comparison of step d), the presence of lungcancer.

According to another aspect, the invention relates to an in vitro methodfor diagnosing metastasized lung cancer in a subject, said methodcomprising the steps of:

a) contacting biological sample from said subject with at least oneprogastrin-binding molecule, and

b) detecting the binding of said progastrin-binding molecule toprogastrin in said sample, wherein said binding indicates the presenceof metastasized lung cancer in said subject.

In a preferred embodiment, the present invention relates to a method forthe in vitro diagnosis of metastasized lung cancer in a subject, from abiological sample of said subject, comprising the steps of:

a) contacting said biological sample with at least oneprogastrin-binding molecule,

b) determining by a biochemical assay the level or concentration ofprogastrin in said biological sample, wherein a concentration ofprogastrin of at least 10 pM higher in said biological sample isindicative of the presence of metastasized lung cancer in said subject.

In a more particular embodiment of a method according to the invention,a concentration of progastrin of at least 10 pM, at least 20 pM, atleast 30 pM, at least 40 pM or at least 50 pM in said biological sampleis indicative of the presence of metastasized lung cancer in saidsubject.

In a more preferred embodiment, the method of the invention comprisesthe further steps of:

a) determining a reference concentration of progastrin in a referencesample,

b) comparing the concentration of progastrin in said biological samplewith said reference concentration of progastrin,

c) diagnosing, from the comparison of step d), the presence ofmetastasized lung cancer.

In a particular embodiment, the present invention relates to a methodfor the in vitro diagnosis of lung cancer in a subject, comprising thedetermination of the concentration of progastrin in a biological sampleand comparing said value obtained to the concentration of progastrin ina reference sample.

In a more particular embodiment, in a method for the diagnosis of lungcancer according to the present invention, the biological sample of saidsubject is contacted with at least one progastrin-binding molecule,wherein said progastrin-binding molecule is an antibody, or anantigen-binding fragment thereof.

The expression “evaluation of a risk of the presence of lung cancer in asubject” designates the determination of a relative probability for agiven subject to suffer from lung cancer, when compared to a referencesubject or value. A method according to the invention represents a toolin the evaluation of said risk, in combination with other methods orindicators such as clinical examination, biopsy and determination of thelevel of a known biomarker of lung cancer.

The expression “in vitro diagnosis” means to determine if a subject issuffering from a particular affection. It is known that the diagnosis oflung cancer involves at least a clinical observation of the symptoms ofsaid subject, such as e.g., low-dose helical computed tomography (CT)scanning. Although some biomarkers were identified in the discoveryphase, it is still a major challenge to transfer them into the clinic,mostly because of the lack of a systematic evaluation process (Li et al,Neoplasma. 2012, 59(5): 500-507).

Therefore, a method for the in vitro diagnosis of lung cancer, accordingto the present invention can be considered as a tool within a diagnosisprocess.

The expression “lung cancer” designates a cancer that originates intissues of the lung, usually in the cells lining air passages. A “lungcancer” as used herein encompasses in particular small cell lung cancer(SCLC), including small cell carcinoma and combined small cellcarcinoma, and non-small cell lung cancers (NSCLC), including squamouscell carcinoma, large cell carcinoma, and adenocarcinoma. Other types oflung cancer such as adenoid cystic carcinomas, lymphomas, and sarcomas,as well as benign lung tumors such as hamartomas are also included inthe lung cancers as used herein.

The term “progastrin” designates the mammalian progastrin peptide, andparticularly human progastrin. For the avoidance of doubt, without anyspecification, the expression “human progastrin” refers to the human PGof sequence SEQ ID No. 1. Human progastrin comprises notably aN-terminus and a C-terminus domains which are not present in thebiologically active gastrin hormone forms mentioned above. Preferably,the sequence of said N-terminus domain is represented by SEQ ID NO. 2.In another preferred embodiment, the sequence of said C-terminus domainis represented by SEQ ID NO. 3.

The determination of the concentration of progastrin, in a methodaccording to the invention, is performed by any method known by oneskilled in the art of biochemistry.

Preferably, determining the levels of progastrin in a sample includescontacting said sample with a progastrin-binding molecule and measuringthe binding of said progastrin-binding molecule to progastrin.

When expression levels are measured at the protein level, it may benotably performed using specific progastrin-binding molecules, such ase.g., antibodies, in particular using well known technologies such ascell membrane staining using biotinylation or other equivalenttechniques followed by immunoprecipitation with specific antibodies,western blot, ELISA or ELISPOT, enzyme-linked immunosorbant assays(ELISA), radioimmunoassays (RIA), immunohistochemistry (IHC),immunofluorescence (IF), antibodies microarrays, or tissue microarrayscoupled to immunohistochemistry. Other suitable techniques include FRETor BRET, single cell microscopic or histochemistry methods using singleor multiple excitation wavelength and applying any of the adaptedoptical methods, such as electrochemical methods (voltametry andamperometry techniques), atomic force microscopy, and radio frequencymethods, e.g. multipolar resonance spectroscopy, confocal andnon-confocal, detection of fluorescence, luminescence,chemiluminescence, absorbance, reflectance, transmittance, andbirefringence or refractive index (e.g., surface plasmon resonance,ellipsometry, a resonant mirror method, a grating coupler waveguidemethod or interferometry), cell ELISA, flow cytometry, radioisotopic,magnetic resonance imaging, analysis by polyacrylamide gelelectrophoresis (SDS-PAGE); HPLC-Mass Spectroscopy; LiquidChromatography/Mass Spectrometry/Mass Spectrometry (LC-MS/MS)). Allthese techniques are well known in the art and need not be furtherdetailed here. These different techniques can be used to measure theprogastrin levels.

Said method may in particular be chosen among: a method based onimmuno-detection, a method based on western blot, a method based on massspectrometry, a method based on chromatography, and a method based onflow cytometry. Although any suitable means for carrying out the assaysare included within the invention, methods such as FACS, ELISA, RIA,western-blot and IHC are particularly useful for carrying out the methodof the invention.

In a more particular embodiment, a method for the in vitro diagnosis oflung cancer according to the invention comprises contacting a biologicalsample from a subject with a progastrin binding molecule using animmunoenzymatic assay, preferably based on techniques chosen among RIAand ELISA.

A “biological sample” as used herein is a sample of biological tissue orfluid that contains nucleic acids or polypeptides, e.g., of a lungcancer protein, polynucleotide or transcript. Such a sample must allowfor the determination of the expression levels of progastrin. Progastrinis known to be a secreted protein. Preferred biological samples for thedetermination of the level of the progastrin protein thus includebiological fluids. A “biological fluid” as used herein means any fluidthat includes material of biological origin. Preferred biological fluidsfor use in the present invention include bodily fluids of an animal,e.g. a mammal, preferably a human subject. The bodily fluid may be anybodily fluid, including but not limited to blood, plasma, serum, lymph,cerebrospinal fluid (CSF), saliva, sweat and urine. Preferably, saidpreferred liquid biological samples include samples such as a bloodsample, a plasma sample, or a serum sample. More preferably, thebiological sample is a blood sample. Indeed, such a blood sample may beobtained by a completely harmless blood collection from the patient andthus allows for a non-invasive assessment of the risks that the subjectwill develop a tumor.

A “biological sample” as used herein also includes a solid cancer sampleof the patient to be tested, when the cancer is a solid cancer. Suchsolid cancer sample allows the skilled person to perform any type ofmeasurement of the level of the biomarker of the invention. In somecases, the methods according to the invention may further comprise apreliminary step of taking a solid cancer sample from the patient. By a“solid cancer sample”, it is referred to a tumor tissue sample. Even ina cancerous patient, the tissue which is the site of the tumor stillcomprises non tumor healthy tissue. The “cancer sample” should thus belimited to tumor tissue taken from the patient. Said “cancer sample” maybe a biopsy sample or a sample taken from a surgical resection therapy.

A biological sample is typically obtained from a eukaryotic organism,most preferably a mammal, or a bird, reptile, or fish. Indeed, a“subject” which may be subjected to the method described herein may beany of mammalian animals including human, dog, cat, cattle, goat, pig,swine, sheep and monkey; or a bird; reptile; or fish. Preferably, asubject is a human being; a human subject may be known as a “patient”.

By “obtaining a biological sample,” it is herein meant to obtain abiological sample for use in methods described in this invention. Mostoften, this will be done by removing a sample of cells from an animal,but can also be accomplished by using previously isolated cells (e.g.,isolated by another person, at another time, and/or for anotherpurpose), or by performing the methods of the invention in vivo.Archival tissues, having treatment or outcome history, will beparticularly useful.

This sample may be obtained and if necessary prepared according tomethods known to a person skilled in the art. In particular, it is wellknown in the art that the sample should be taken from a fasting subject.

The determination of the concentration of progastrin relates to thedetermination of the quantity of progastrin in known volume of a sample.The concentration of progastrin may be expressed relatively to areference sample, for example as a ratio or a percentage. Theconcentration may also be expressed as the intensity or localization ofa signal, depending on the method used for the determination of saidconcentration. Preferably, the concentration of a compound in a sampleis expressed after normalization of the total concentration of relatedcompounds in said sample, for example the level or concentration of aprotein is expressed after normalization of the total concentration ofproteins in the sample.

Preferably, the risk that said subject suffers from lung cancer isdetermined by comparing the level of progastrin measured in saidbiological sample with a reference level.

The term “reference level”, as used herein, refers to the expressionlevel of the lung cancer marker under consideration, i.e. progastrin, ina reference sample. A “reference sample”, as used herein, means a sampleobtained from subjects, preferably two or more subjects, known to befree of the disease or, alternatively, from the general population. Thesuitable reference expression levels of progastrin can be determined bymeasuring the expression levels of said marker in several suitablesubjects, and such reference levels can be adjusted to specific subjectpopulations. The reference value or reference level can be an absolutevalue; a relative value; a value that has an upper or a lower limit; arange of values; an average value; a median value, a mean value, or avalue as compared to a particular control or baseline value. A referencevalue can be based on an individual sample value such as, for example, avalue obtained from a sample from the subject being tested, but at anearlier point in time. The reference value can be based on a largenumber of samples, such as from population of subjects of thechronological age matched group, or based on a pool of samples includingor excluding the sample to be tested.

Advantageously, a “reference level” is a predetermined progastrin level,obtained from a biological sample from a subject with a known particularstatus as regards cancer. In particular embodiments, the reference levelused for comparison with the test sample in step (b) may have beenobtained from a biological sample from a healthy subject, or from abiological sample from a subject suffering from cancer; it is understoodthat the reference expression profile can also be obtained from a poolof biological samples of healthy subjects or from a pool of samples fromsubjects having cancer.

In a particular embodiment of the method of the invention, the referencesample is collected from subjects exempt from any cancer, and preferablyfrom any pathology. It is to be understood that, according to the natureof the biological sample collected from a patient, the reference samplewill be a biological sample of the same nature of said biologicalsample.

The level of progastrin is determined in the present method bydetermining the amount of progastrin which is bound by aprogastrin-binding molecule, preferably by an antibody recognisingprogastrin.

By “progastrin-binding molecule”, it is herein referred to any moleculethat binds progastrin, but does not bind gastrin-17 (G17), gastrin-34(G34), glycine-extended gastrin-17 (G17-Gly), or glycine-extendedgastrin-34 (G34-Gly). The progastrin-binding molecule of the presentinvention may be any progastrin-binding molecule, such as, for instance,an antibody molecule or a receptor molecule. Preferably, theprogastrin-binding molecule is an anti-progastrin antibody or anantigen-binding fragment thereof.

According to a particular embodiment, the present invention relates toan in vitro diagnosis method of a lung cancer comprising thedetermination of the concentration of progastrin in a biological samplefrom a subject, wherein said subject exhibits at least one clinicalsymptom of lung cancer.

According to another particular embodiment, the present inventionrelates to an in vitro diagnosis method of a lung cancer comprising thedetermination of the concentration of progastrin in a biological samplefrom a subject, wherein said subject exhibits at least one clinicalsymptom of cancer and/or of metastasis.

By “binding”, “binds”, or the like, it is intended that the antibody, orantigen binding fragment thereof, forms a complex with an antigen which,under physiologic conditions, is relatively stable. Methods fordetermining whether two molecules bind are well known in the art andinclude, for example, equilibrium dialysis, surface plasmon resonance,and the like. In a particular embodiment, said antibody, orantigen-binding fragment thereof, binds to progastrin with an affinitythat is at least two-fold greater than its affinity for binding to anon-specific molecule such as BSA or casein. In a more particularembodiment, said antibody, or antigen-binding fragment thereof, bindsonly to progastrin.

In a particular embodiment, in a method for the diagnosis of lung canceraccording to the invention, a biological sample from the subject iscontact with at least one progastrin-binding molecule, wherein theaffinity of said molecule for progastrin is of at least 100 nM, at least90 nM, at least 80 nM, at least 70 nM, at least 60 nM, at least 50 nM,at least 40 nM, at least 30 nM, at least 20 nM, at least 10 nM, at least5 nM, at least 1 nM, at least 100 pM, at least 10 pM, or at least 1 pM,as determined by a method such as above-described.

In a particular embodiment, the present invention relates to a methodfor the diagnosis of lung cancer, comprising the detection of theconcentration of progastrin in a biological sample from a subject,wherein said biological sample is contacted with an anti-hPG antibody,or an antigen-binding fragment thereof.

The term “antibody” as used herein is intended to include polyclonal andmonoclonal antibodies. An antibody (or “immunoglobulin”) consists of aglycoprotein comprising at least two heavy (H) chains and two light (L)chains inter-connected by disulfide bonds. Each heavy chain comprises aheavy chain variable region (or domain) (abbreviated herein as HCVR orVH) and a heavy chain constant region. The heavy chain constant regioncomprises three domains, CH1, CH2 and CH3. Each light chain comprises alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region comprisesone domain, CL. The VH and VL regions can be further subdivided intoregions of hypervariability, termed “complementarity determiningregions” (CDR) or “hypervariable regions”, which are primarilyresponsible for binding an epitope of an antigen, and which areinterspersed with regions that are more conserved, termed frameworkregions (FR). Method for identifying the CDRs within light and heavychains of an antibody and determining their sequence are well known tothe skilled person. For the avoidance of doubt, in the absence of anyindication in the text to the contrary, the expression CDRs means thehypervariable regions of the heavy and light chains of an antibody asdefined by IMGT, wherein the IMGT unique numbering provides astandardized delimitation of the framework regions and of thecomplementary determining regions, CDR1-IMGT: 27 to 38, CDR2.

The IMGT unique numbering has been defined to compare the variabledomains whatever the antigen receptor, the chain type, or the species[Lefranc M. -P., Immunology Today 18, 509 (1997)/Lefranc M. -P., TheImmunologist, 7, 132-136 (1999)/Lefranc, M. -P., Pommié, C., Ruiz, M.,Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. andLefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. In the IMGT uniquenumbering, the conserved amino acids always have the same position, forinstance cystein 23 (1st-CYS), tryptophan 41 (CONSERVED-TRP),hydrophobic amino acid 89, cystein 104 (2nd-CYS), phenylalanine ortryptophan 118 (J-PHE or J-TRP). The IMGT unique numbering provides astandardized delimitation of the framework regions (FR1-IMGT: positions1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to128) and of the complementarity determining regions: CDR1-IMGT: 27 to38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117. As gaps representunoccupied positions, the CDR-IMGT lengths (shown between brackets andseparated by dots, e.g. [8.8.13]) become crucial information. The IMGTunique numbering is used in 2D graphical representations, designated asIMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics,53, 857-883 (2002)/Kaas, Q. and Lefranc, M.-P., Current Bioinformatics,2, 21-30 (2007)], and in 3D structures in IMGT/3Dstructure-DB [Kaas, Q.,Ruiz, M. and Lefranc, M. -P., T cell receptor and MHC structural data.Nucl. Acids. Res., 32, D208-D210 (2004)].

Each VH and VL is composed of three CDRs and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g. effector cells) and the first component (Clq) ofthe classical complement system. Antibodies can be of different isotypes(namely IgA, IgD, IgE, IgG or IgM).

In a particular embodiment, said progastrin-binding antibody, or anantigen-binding fragment thereof, is selected from the group consistingof: polyclonal antibodies, monoclonal antibodies, chimeric antibodies,single chain antibodies, camelized antibodies, IgA1 antibodies, IgA2antibodies, IgD antibodies, IgE antibodies, IgG1 antibodies, IgG2antibodies, IgG3 antibodies, IgG4 antibodies and IgM antibodies.

A “polyclonal antibody” is an antibody which was produced among or inthe presence of one or more other, non-identical antibodies. In general,polyclonal antibodies are produced from a B-lymphocyte in the presenceof several other B-lymphocytes producing non-identical antibodies.Usually, polyclonal antibodies are obtained directly from an immunizedanimal.

The term “monoclonal antibody” designates an antibody arising from anearly homogeneous antibody population, wherein population comprisesidentical antibodies except for a few possible naturally-occurringmutations which can be found in minimal proportions. A monoclonalantibody arises from the growth of a single cell clone, such as ahybridoma, and is characterized by heavy chains of one class andsubclass, and light chains of one type.

By the expression “antigen-binding fragment” of an antibody, it isintended to indicate any peptide, polypeptide, or protein retaining theability to bind to the target (also generally referred to as antigen) ofthe said antibody, generally the same epitope, and comprising an aminoacid sequence of at least 5 contiguous amino acid residues, at least 10contiguous amino acid residues, at least 15 contiguous amino acidresidues, at least 20 contiguous amino acid residues, at least 25contiguous amino acid residues, at least 40 contiguous amino acidresidues, at least 50 contiguous amino acid residues, at least 60contiguous amino residues, at least 70 contiguous amino acid residues,at least 80 contiguous amino acid residues, at least 90 contiguous aminoacid residues, at least 100 contiguous amino acid residues, at least 125contiguous amino acid residues, at least 150 contiguous amino acidresidues, at least 175 contiguous amino acid residues, or at least 200contiguous amino acid residues, of the amino acid sequence of theantibody.

In a particular embodiment, the said antigen-binding fragment comprisesat least one CDR of the antibody from which it is derived. Still in apreferred embodiment, the said antigen binding fragment comprises 2, 3,4 or 5 CDRs, more preferably the 6 CDRs of the antibody from which it isderived.

The “antigen-binding fragments” can be selected, without limitation, inthe group consisting of Fv, scFv (sc for single chain), Fab, F(ab′)₂,Fab′, scFv-Fc fragments or diabodies, or fusion proteins with disorderedpeptides such as XTEN (extended recombinant polypeptide) or PAS motifs,or any fragment of which the half-life time would be increased bychemical modification, such as the addition of poly(alkylene) glycolsuch as poly(ethylene) glycol (“PEGylation”) (pegylated fragments calledFv-PEG, scFv-PEG, Fab-PEG, F(ab′)₂-PEG or Fab′-PEG) (“PEG” forPoly(Ethylene) Glycol), or by incorporation in a liposome, saidfragments having at least one of the characteristic CDRs of the antibodyaccording to the invention. Preferably, said “antigen-binding fragments”will be constituted or will comprise a partial sequence of the heavy orlight variable chain of the antibody from which they are derived, saidpartial sequence being sufficient to retain the same specificity ofbinding as the antibody from which it is descended and a sufficientaffinity, preferably at least equal to 1/100, in a more preferred mannerto at least 1/10, of the affinity of the antibody from which it isdescended, with respect to the target.

In another particular embodiment, in a method for the diagnosis of lungcancer according to the invention, a biological sample from a subject iscontacted with an antibody binding to progastrin, wherein said antibodyhas been obtained by an immunization method known by a person skilled inthe art, wherein using as an immunogen a peptide which amino acidsequence comprises the totality or a part of the amino-acid sequence ofprogastrin. More particularly, said immunogen comprises a peptide chosenamong:

a peptide which amino acid sequence comprises, or consists of, the aminoacid sequence of full length progastrin, and particularly full lengthhuman progastrin of SEQ ID No 1,

a peptide which amino acid sequence corresponds to a part of the aminoacid sequence of progastrin, and particularly full length humanprogastrin of SEQ ID No 1,

a peptide which amino acid sequence corresponds to a part or to thewhole amino acid sequence of the N-terminal part of progastrin, and inparticular peptides comprising, or consisting of, the amino acidsequence: SWKPRSQQPDAPLG (SEQ ID No 2), and

a peptide which amino acid sequence corresponds to a part or to thewhole amino acid sequence of the C-terminal part of progastrin, and inparticular peptides comprising, or consisting of, the amino acidsequence: QGPWLEEEEEAYGWMDFGRRSAEDEN (SEQ ID No 3),

a peptide which amino acid sequence corresponds to a part of the aminoacid sequence of the C-terminal part of progastrin, and in particularpeptides comprising the amino acid sequence FGRRSAEDEN (SEQ ID No 40)corresponding to amino acids 71-80 of progastrin

The skilled person will realize that such immunization may be used togenerate either polyclonal or monoclonal antibodies, as desired. Methodsfor obtaining each of these types of antibodies are well known in theart. The skilled person will thus easily select and implement a methodfor generating polyclonal and/or monoclonal antibodies against any givenantigen.

Examples of monoclonal antibodies which were generated by using animmunogen comprising the amino-acid sequence “SWKPRSQQPDAPLG”,corresponding to the amino acid sequence 1-14 of human progastrin(N-terminal extremity) include, but are not restricted to, monoclonalantibodies designated as: mAb3, mAb4, mAb16, and mAb19 and mAb20, asdescribed in the following Table 1 to Table 4. Other monoclonalantibodies have been described, although it is not clear whether theseantibodies actually bind progastrin (WO 2006/032980). Experimentalresults of epitope mapping show that mAb3, mAb4, mAb16, and mAb19 andmAb20 do specifically bind an epitope within said hPG N-terminal aminoacid sequence. Polyclonal antibodies recognizing specifically an epitopewithin the N-terminus of progastrin represented by SEQ ID NO. 2, havebeen described in the art (see e.g, WO 2011/083088).

TABLE 1 Hybridoma Amino acid deposit mAb sequences SEQ ID No 6B5B11C10mAb3 VH CDR 1 GYIFTSYW SEQ ID No 4 VH CDR 2 FYPGNSDS SEQ ID No 5VH CDR 3 TRRDSPQY SEQ ID No 6 VL CDR 1 QSIVHSNGNTY SEQ ID No 7 VL CDR 2KVS SEQ ID No 8 VL CDR 3 FQGSHVPFT SEQ ID No 9 mVH 3EVQLQQSGTVLARPGASVKMSCK SEQ ID No 41 ASGYIFTSYWVHWVKQRPGQGLEWIGGFYPGNSDSRYNQKFKGKAT LTAVTSASTAYMDLSSLTNEDSAV YFCTRRDSPQYWGQGTTLTVSSmVL 3 DVLMTQTPLSLPVSLGDQASISCR SEQ ID No 42 SSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRLEAEDLGVYYCFQG SHVPFTFGGGTKLEIKhuVH 3 QVQLVQSGAEVKKPGASVKVSCK SEQ ID No 53 ASGYIFTSYWVHWVRQAPGQRLEWMGGFYPGNSDSRYSQKFQGRV TITRDTSASTAYMELSSLRSEDTAV YYCTRRDSPQYWGQGTLVTVSShuVL 3 DVVMTQSPLSLPVTLGQPASISCR SEQ ID No 54 SSQSIVHSNGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDVGVYYCFQG SHVPFTFGGGTKVEIK

TABLE 2 Hybridoma Amino acid deposit mAb sequences SEQ ID No 20D2C3G2mAb4 VH CDR 1 GYTFSSW SEQ ID No 10 VH CDR 2 FLPGSGST SEQ ID No 11VH CDR 3 ATDGNYDWFAY SEQ ID No 12 VL CDR 1 QSLVHSSGVTY SEQ ID No 13VL CDR 2 KVS SEQ ID No 14 VL CDR 3 SQSTHVPPT SEQ ID No 15 mVH 4QVQLQQSGAELMKPGASVKISCK SEQ ID No 43 ATGYTFSSSWIEWLKQRPGHGLEWIGEFLPGSGSTDYNEKFKGKATF TADTSSDTAYMLLSSLTSEDSAVY YCATDGNYDWFAYWGQGTLVTVSA mVL 4 DLVMTQTPLSLPVSLGDQASISCR SEQ ID No 44 SSQSLVHSSGVTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDLGVYFCSQS THVPPTFGSGTKLEIKhuVH 4 QVQLVQSGAEVKKPGASVKVSCK SEQ ID No 55 ASGYTFSSSWMHWVRQAPGQGLEWMGIFLPGSGSTDYAQKFQGRV TMTRDTSTSTVYMELSSLRSEDTA VYYCATDGNYDWFAYWGQGTLVTVSS huVL 4 DIVMTQTPLSLSVTPGQPASISCKS SEQ ID No 56SQSLVHSSGVTYLYWYLQKPGQS PQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQS THVPPTFGQGTKLEIK

TABLE 3 Hybridoma Amino acid deposit mAb sequences SEQ ID No 1E9D9B6mAb16 VH CDR 1 GYTFTSYY SEQ ID No 16 VH CDR 2 INPSNGGT SEQ ID No 17VH CDR 3 TRGGYYPFDY SEQ ID No 18 VL CDR 1 QSLLDSDGKTY SEQ ID No 19VL CDR 2 LVS SEQ ID No 20 VL CDR 3 WQGTHSPYT SEQ ID No 21 mVH 16QVQLQQSGAELVKPGASVKLSCK SEQ ID No 45 ASGYTFTSYYMYWVKQRPGQGLEWIGEINPSNGGTNFNEKFKSKATL TVDKSSSTAYMQLSSLTSEDSAVYYCTRGGYYPFDYWGQGTTLTVSS mVL 16 DVVMTQTPLTLSVTIGRPASISCKS SEQ ID No 46SQSLLDSDGKTYLYWLLQRPGQS PKRLIYLVSELDSGVPDRITGSGSGTDFTLKISRVEAEDLGVYYCWQG THSPYTFGGGTKLEIK huVH 16aQVQLVQSGAEVKKPGASVKVSCK SEQ ID No 57 ASGYTFTSYYMYWVRQAPGQGLEWMGIINPSNGGTSYAQKFQGRVT MTRDTSTSTVYMELSSLRSEDTAV YYCTRGGYYPFDYWGQGTTVTVSS huVH 16b QVQLVQSGAEVKKPGASVKVSCK SEQ ID No 58 ASGYTFTSYYMHWVRQAPGQGLEWMGIINPSNGGTSYAQKFQGRV TMTRDTSTSTVYMELSSLRSEDTA VYYCTRGGYYPFDYWGQGTTVTVSS huVH 16c QVQLVQSGAEVKKPGASVKVSCK SEQ ID No 59ASGYTFTSYYMYWVRQAPGQGLE WMGEINPSNGGTNYAQKFQGRV TMTRDTSTSTVYMELSSLRSEDTAVYYCTRGGYYPFDYWGQGTTVT VSS huVL 16a DVVMTQSPLSLPVTLGQPASISCRSEQ ID No 60 SSQSLLDSDGKTYLYWFQQRPGQ SPRRLIYLVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCWQ GTHSPYTFGQGTKLEIK huVL 16bDVVMTQSPLSLPVTLGQPASISCR SEQ ID No 61 SSQSLLDSDGKTYLNWFQQRPGQSPRRLIYLVSNRDSGVPDRFSGSGS GTDFTLKISRVEAEDVGVYYCWQ GTHSPYTFGQGTKLEIKhuVL 16c DVVMTQSPLSLPVTLGQPASISCR SEQ ID No 62 SSQSLLDSDGKTYLYWFQQRPGQSPRRLIYLVSERDSGVPDRFSGSGS GTDFTLKISRVEAEDVGVYYCWQ GTHSPYTFGQGTKLEIK

TABLE 4 Hybridoma Amino acid deposit mAb sequences SEQ ID No 1B3B4F11mAb19 VH CDR 1 GYSITSDYA SEQ ID No 22 VH CDR 2 ISFSGYT SEQ ID No 23VH CDR 3 AREVNYGDSYHFDY SEQ ID No 24 VL CDR 1 SQHRTYT SEQ ID No 25VL CDR 2 VKKDGSH SEQ ID No 26 VL CDR 3 GVGDAIKGQSVFV SEQ ID No 27 mVH 19DVQLQESGPGLVKPSQSLSLTCTV SEQ ID No 47 TGYSITSDYAWNWIRQFPGNKLEWMGYISFSGYTSYNPSLKSRISVTR DTSRNQFFLQLTSVTTEDTATYYCAREVNYGDSYHFDYWGQGTIVTV SS mVL 19 QLALTQSSSASFSLGASAKLTCTLS SEQ ID No 48SQHRTYTIEWYQQQSLKPPKYVM EVKKDGSHSTGHGIPDRFSGSSSGADRYLSISNIQPEDEAIYICGVGDAI KGQSVFVFGGGTKVTVL huVH 19aQVQLQESGPGLVKPSQTLSLTCT SEQ ID No 63 VSGYSITSDYAWNWIRQHPGKGLEWIGYISFSGYTYYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVYYCAREVNYGDSYHFDYWGQGTLV TVSS huVH 19b QVQLQESGPGLVKPSQTLSLTCTSEQ ID No 64 VSGYSITSDYAWSWIRQHPGKGLE WIGYISFSGYTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC AREVNYGDSYHFDYWGQGTLVT VSS huVH 19cQVQLQESGPGLVKPSQTLSLTCT SEQ ID No 65 VSGYSITSDYAWNWIRQHPGKGLEWIGYISFSGYTSYNPSLKSRVTIS VDTSKNQFSLKLSSVTAADTAVYYCAREVNYGDSYHFDYWGQGTLV TVSS huVL 19a QLVLTQSPSASASLGASVKLTCTLSEQ ID No 66 SSQHRTYTIEWHQQQPEKGPRYL MKVKKDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDEADYYCGVG DAIKGQSVFVFGGGTKVEIK huVL 19bQLVLTQSPSASASLGASVKLTCTL SEQ ID No 67 SSQHRTYTIAWHQQQPEKGPRYLMKVKKDGSHSKGDGIPDRFSGSSS GAERYLTISSLQSEDEADYYCGVG DAIKGQSVFVFGGGTKVEIKhuVL 19c QLVLTQSPSASASLGASVKLTCTL SEQ ID No 68 SSQHRTYTIEWHQQQPEKGPRYLMEVKKDGSHSKGDGIPDRFSGSSS GAERYLTISSLQSEDEADYYCGVG DAIKGQSVFVFGGGTKVEIK

Examples of monoclonal antibodies that can be generated by using animmunogen comprising the amino-acid sequence“QGPWLEEEEEAYGWMDFGRRSAEDEN”, (C-terminal part of progastrin)corresponding to the amino acid sequence 55-80 of human progastrininclude, but are not restricted to antibodies designated as: mAb8 andmAb13 in the following Table 5 and 6. Experimental results of epitopemapping show that mAb13 do specifically bind an epitope within said hPGC-terminal amino acid sequence. Another example of a monoclonal antibodythat can thus be generated by is the antibody Mab14, produced byhybridoma 2H9F4B7, described in WO 2011/083088. Hybridoma 2H9F4B7 wasdeposited under the Budapest Treaty at the CNCM, Institut Pasteur, 25-28rue du Docteur Roux, 75724 Paris CEDEX 15, France, on 27 Dec. 2016,under reference I-5158 (see WO 2017/114973).

TABLE 5 Hybridoma Amino acid deposit mAb sequences SEQ ID No 1C10D3B9mAb8 VH CDR 1 GFTFTTYA SEQ ID No 28 VH CDR 2 ISSGGTYT SEQ ID No 29VH CDR 3 ATQGNYSLDF SEQ ID No 30 VL CDR 1 KSLRHTKGITF SEQ ID No 31VL CDR 2 QMS SEQ ID No 32 VL CDR 3 AQNLELPLT SEQ ID No 33 mVH 8EVQLVESGGGLVKPGGSLRLSC SEQ ID No 49 AASGFTFTTYAMSWVRQAPGKGLEWVATISSGGTYTYYADSVK GRFTISRDNAKNSLYLQMNSLRA EDTAVYYCATQGNYSLDFWGQGTTVTVSS mVL 8 DIVMTQSPLSLPVTPGEPASISCR SEQ ID No 50SSKSLRHTKGITFLYWYLQKPGQ SPQLLIYQMSNLASGVPDRFSSS GSGTDFTLKISRVEAEDVGVYYCAQNLELPLTFGGGTKVEIK VH hZ8CV1 EVQLVESGGGLVKPGGSLRLSC SEQ ID No 69AASGFTFTTYAMSWVRQAPGK GLEWVSSISSGGTYTYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCATQGNYSLDFWGQG TTVTVSS VL hZ8CV1 DIVMTQSPLSLPVTPGEPASISCRSEQ ID No 70 SSKSLRHTKGITFLYWYLQKPGQ SPQLLIYQMSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC AQNLELPLTFGGGTKVEIK VH hZ8CV2EVQLVESGGGLVKPGGSLRLSC SEQ ID No 71 AASGFTFTTYAMSWVRQAPGKGLEWVATISSGGTYTYYADSVK GRFTISRDNAKNSLYLQMNSLRA EDTAVYYCATQGNYSLDFWGQGTTVTVSS VL hZ8CV2 DIVMTQSPLSLPVTPGEPASISCR SEQ ID No 72SSKSLRHTKGITFLYWYLQKPGQ SPQLLIYQMSNLASGVPDRFSSS GSGTDFTLKISRVEAEDVGVYYCAQNLELPLTFGGGTKVEIK CH hZ8CV2 EVQLVESGGGLVKPGGSLRLSC SEQ ID No 73AASGFTFTTYAMSWVRQAPGK GLEWVATISSGGTYTYYADSVK GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCATQGNYSLDFWGQ GTTVTVSSASTKGPSVFPLAPSS KSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ DWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K CL hZ8CV2 DIVMTQSPLSLPVTPGEPASISCR SEQ ID No 74SSKSLRHTKGITFLYWYLQKPGQ SPQLLIYQMSNLASGVPDRFSSS GSGTDFTLKISRVEAEDVGVYYCAQNLELPLTFGGGTKVEIKRTVA APSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC

TABLE 6 Hybridoma Amino acid deposit mAb sequences SEQ ID No 2C6C3C7mAb13 VH CDR 1 GFIFSSYG SEQ ID No 34 VH CDR 2 INTFGDRT SEQ ID No 35VH CDR 3 ARGTGTY SEQ ID No 36 VL CDR 1 QSLLDSDGKTY SEQ ID No 37 VL CDR 2LVS SEQ ID No 38 VL CDR 3 WQGTHFPQT SEQ ID No 39 mVH 13EVQLVESGGGLVQPGGSLKLSC SEQ ID No 51 AASGFIFSSYGMSWVRQSPDRRLELVASINTFGDRTYYPDSVKGRF TISRDNAKNTLYLQMTSLKSEDT AIYYCARGTGTYWGQGTTLTVS SmVL 13 DVVLTQTPLTLSVTIGQPASISCK SEQ ID No 52 SSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTG SGSGTDFTLKISRVEAEDLGVYY CWQGTHFPQTFGGGTKLEIKhuVH 13a EVQLVESGGGLVQPGGSLRLSC SEQ ID No 75 AASGFIFSSYGMSWVRQAPGKGLEWVANINTFGDRTYYVDSVKG RFTISRDNAKNSLYLQMNSLRAE DTAVYYCARGTGTYWGQGTLVTVSS huVH 13b EVQLVESGGGLVQPGGSLRLSC SEQ ID No 76 AASGFIFSSYGMSWVRQAPGKGLEWVASINTFGDRTYYVDSVKG RFTISRDNAKNSLYLQMNSLRAE DTAVYYCARGTGTYWGQGTLVTVSS huVL 13a DVVMTQSPLSLPVTLGQPASISC SEQ ID No 77RSSQSLLDSDGKTYLNWFQQRP GQSPRRLIYLVSNRDSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPQTFGGGTKVEIK huVL 13b DVVMTQSPLSLPVTLGQPASISC SEQ ID No 78RSSQSLLDSDGKTYLNWFQQRP GQSPRRLIYLVSKRDSGVPDRFS GSGSGTDFTLKISRVEAEDVGVYYCWQGTHFPQTFGGGTKVEIK

Other examples include anti-hPG monoclonal and/or polyclonal antibodiesgenerated by using an immunogen comprising an amino acid sequence of SEQID No 40.

In a more particular embodiment, in a method according to the inventionsaid biological sample is contacted with an anti-hPG antibody orantigen-binding fragment thereof, wherein said anti-hPG antibody ischosen among N-terminal anti-hPG antibodies and C-terminal anti-hPGantibodies.

The terms “N-terminal anti-hPG antibodies” and “C-terminal anti-hPGantibodies” designate antibodies binding to an epitope comprising aminoacids located in the N-terminal part of hPG or to an epitope comprisingamino acids located in the C-terminal part of hPG, respectively.Preferably, the term “N-terminal anti-hPG antibodies” refers toantibodies binding to an epitope located in a domain of progastrin whosesequence is represented by SEQ ID NO. 2. In another preferredembodiment, the term “C-terminal anti-hPG antibodies” refers toantibodies binding to an epitope located in a domain of progastrin whosesequence is represented by SEQ ID NO. 3.

The term “epitope” refers to a region of an antigen that is bound by anantibody. Epitopes may be defined as structural or functional.Functional epitopes are generally a subset of the structural epitopesand have those amino acids that directly contribute to the affinity ofthe interaction. Epitopes may also be conformational. In certainembodiments, epitopes may include determinants that are chemicallyactive surface groupings of molecules such as amino acids, sugar sidechains, phosphoryl groups, or sulfonyl groups, and, in certainembodiments, may have specific three-dimensional structuralcharacteristics, and/or specific charge characteristics. Thedetermination of the epitope bound by an antibody may be performed byany epitope mapping technique, known by a man skilled in the art. Anepitope may comprise different amino acids which located sequentiallywithin the amino acid sequence of a protein. An epitope may alsocomprise amino acids which are not located sequentially within the aminoacid sequence of a protein.

In a particular embodiment, said antibody is a monoclonal antibodyselected in the group consisting of:

A monoclonal antibody comprising a heavy chain comprising at least one,preferentially at least two, preferentially three, of CDR-H1, CDR-H2 andCDR-H3 of amino acid sequences SEQ ID No 4, 5 and 6, respectively, orsequences with at least 80%, preferably 85%, 90%, 95% and 98% identityafter optimal alignment with sequences SEQ ID No 4, 5 and 6,respectively, and a light chain comprising at least one, preferentiallyat least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 ofamino acid sequences SEQ ID No 7, 8 and 9, respectively, or sequenceswith at least 80%, preferably 85%, 90%, 95% and 98% identity afteroptimal alignment with sequences SEQ ID No 7, 8 and 9, respectively,

A monoclonal antibody comprising a heavy chain comprising at least one,preferentially at least two, preferentially three, of CDR-H1, CDR-H2 andCDR-H3 of amino acid sequences SEQ ID No 10, 11 and 12, respectively, orsequences with at least 80%, preferably 85%, 90%, 95% and 98% identityafter optimal alignment with sequences SEQ ID No 10, 11 and 12,respectively, and a light chain comprising at least one, preferentiallyat least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 ofamino acid sequences SEQ ID No 13, 14 and 15, respectively, or sequenceswith at least 80%, preferably 85%, 90%, 95% and 98% identity afteroptimal alignment with sequences SEQ ID No 13, 14 and 15, respectively,

A monoclonal antibody comprising a heavy chain comprising at least one,preferentially at least two, preferentially three, of CDR-H1, CDR-H2 andCDR-H3 of amino acid sequences SEQ ID No 16, 17 and 18, respectively, orsequences with at least 80%, preferably 85%, 90%, 95% and 98% identityafter optimal alignment with sequences SEQ ID No 16, 17 and 18,respectively, and a light chain comprising at least one, preferentiallyat least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 ofamino acid sequences SEQ ID No 19, 20 and 21, respectively, or sequenceswith at least 80%, preferably 85%, 90%, 95% and 98% identity afteroptimal alignment with sequences SEQ ID No 19, 20 and 21, respectively,

A monoclonal antibody comprising a heavy chain comprising at least one,preferentially at least two, preferentially three, of CDR-H1, CDR-H2 andCDR-H3 of amino acid sequences SEQ ID No 22, 23 and 24, respectively, orsequences with at least 80%, preferably 85%, 90%, 95% and 98% identityafter optimal alignment with sequences SEQ ID No 22, 23 and 24,respectively, and a light chain comprising at least one, preferentiallyat least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 ofamino acid sequences SEQ ID No 25, 26 and 27, respectively, or sequenceswith at least 80%, preferably 85%, 90%, 95% and 98% identity afteroptimal alignment with sequences SEQ ID No 25, 26 and 27, respectively,

A monoclonal antibody comprising a heavy chain comprising at least one,preferentially at least two, preferentially at least three, of CDR-H1,CDR-H2 and CDR-H3 of amino acid sequences SEQ ID No 28, 29 and 30,respectively, or sequences with at least 80%, preferably 85%, 90%, 95%and 98% identity after optimal alignment with sequences SEQ ID No 28, 29and 30, respectively, and a light chain comprising at least one,preferentially at least two, preferentially three, of CDR-L1, CDR-L2 andCDR-L3 of amino acid sequences SEQ ID No 31, 32 and 33, respectively, orsequences with at least 80%, preferably 85%, 90%, 95% and 98% identityafter optimal alignment with sequences SEQ ID No 31, 32 and 33,respectively, and

A monoclonal antibody comprising a heavy chain comprising at least one,preferentially at least two, preferentially three, of CDR-H1, CDR-H2 andCDR-H3 of amino acid sequences SEQ ID No 34, 35 and 36, respectively, orsequences with at least 80%, preferably 85%, 90%, 95% and 98% identityafter optimal alignment with sequences SEQ ID No 34, 35 and 36,respectively, and a light chain comprising at least one, preferentiallyat least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 ofamino acid sequences SEQ ID No 37, 38 and 39, respectively, or sequenceswith at least 80%, preferably 85%, 90%, 95% and 98% identity afteroptimal alignment with sequences SEQ ID No 37, 38 and 39, respectively.

In another embodiment, the antibody is a monoclonal antibody produced bythe hybridoma deposited at the CNCM, Institut Pasteur, 25-28 rue duDocteur Roux, 75724 Paris CEDEX 15, France, on 27 Dec. 2016, underreference 1-5158 (see WO 2017/114973).

In the sense of the present invention, the “percentage identity” or “%identity” between two sequences of nucleic acids or amino acids meansthe percentage of identical nucleotides or amino acid residues betweenthe two sequences to be compared, obtained after optimal alignment, thispercentage being purely statistical and the differences between the twosequences being distributed randomly along their length. The comparisonof two nucleic acid or amino acid sequences is traditionally carried outby comparing the sequences after having optimally aligned them, saidcomparison being able to be conducted by segment or by using an“alignment window”. Optimal alignment of the sequences for comparisoncan be carried out, in addition to comparison by hand, by means ofmethods known by a man skilled in the art.

For the amino acid sequence exhibiting at least 80%, preferably 85%,90%, 95% and 98% identity with a reference amino acid sequence,preferred examples include those containing the reference sequence,certain modifications, notably a deletion, addition or substitution ofat least one amino acid, truncation or extension. In the case ofsubstitution of one or more consecutive or non-consecutive amino acids,substitutions are preferred in which the substituted amino acids arereplaced by “equivalent” amino acids. Here, the expression “equivalentamino acids” is meant to indicate any amino acids likely to besubstituted for one of the structural amino acids without howevermodifying the biological activities of the corresponding antibodies andof those specific examples defined below.

Equivalent amino acids can be determined either on their structuralhomology with the amino acids for which they are substituted or on theresults of comparative tests of biological activity between the variousantibodies likely to be generated.

In a more particular embodiment, said antibody is a monoclonal antibodyselected in the group consisting of:

A monoclonal antibody comprising a heavy chain of amino acid sequenceSEQ ID No 41 and a light chain of amino acid sequence SEQ ID No 42;

A monoclonal antibody comprising a heavy chain of amino acid sequenceSEQ ID No 43 and a light chain of amino acid sequence SEQ ID No 44;

A monoclonal antibody comprising a heavy chain of amino acid sequenceSEQ ID No 45 and a light chain of amino acid sequence SEQ ID No 46;

A monoclonal antibody comprising a heavy chain of amino acid sequenceSEQ ID No 47 and a light chain of amino acid sequence SEQ ID No 48;

A monoclonal antibody comprising a heavy chain of amino acid sequenceSEQ ID No 49 and a light chain of amino acid sequence SEQ ID No 50; and

A monoclonal antibody comprising a heavy chain of amino acid sequenceSEQ ID No 51 and a light chain of amino acid sequence SEQ ID No 52.

In another particular embodiment, the antibody used in the method of theinvention is a humanised antibody.

As used herein, the expression “humanized antibody” means an antibodythat contains CDR regions derived from an antibody of nonhuman origin,the other parts of the antibody molecule being derived from one orseveral human antibodies. In addition, some of the skeleton segmentresidues (called FR for framework) can be modified to preserve bindingaffinity, according to techniques known by a man skilled in the art(Jones et al., Nature, 321:522-525, 1986). The goal of humanisation is areduction in the immunogenicity of a xenogenic antibody, such as amurine antibody, for introduction into a human, while maintaining thefull antigen binding affinity and specificity of the antibody.

The humanized antibodies of the invention or fragments of same can beprepared by techniques known to a person skilled in the art (such as,for example, those described in the documents Singer et al., J. Immun.,150:2844-2857, 1992). Such humanized antibodies are preferred for theiruse in methods involving in vitro diagnoses or preventive and/ortherapeutic treatment in vivo. Other humanization techniques are alsoknown to the person skilled in the art. Indeed, Antibodies can behumanized using a variety of techniques including CDR-grafting (EP 0 451261; EP 0 682 040; EP 0 939 127; EP 0 566 647; U.S. Pat. Nos. 5,530,101;6,180,370; 5,585,089; 5,693,761; 5,639,641; 6,054,297; 5,886,152; and5,877,293), veneering or resurfacing (EP 0 592 106; EP 0 519 596; PadlanE. A., 1991, Molecular Immunology 28(4/5): 489-498; Studnicka G. M. etal., 1994, Protein Engineering 7(6): 805-814; Roguska M. A. et al.,1994, Proc. Natl. Acad. ScL U.S.A., 91:969-973), and chain shuffling(U.S. Pat. No. 5,565,332). Human antibodies can be made by a variety ofmethods known in the art including phage display methods. See also U.S.Pat. Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; andinternational patent application publication numbers WO 98/46645, WO98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO91/10741.

In a more particular embodiment, said antibody is a humanized antibodyselected in the group consisting of:

A humanized antibody comprising a heavy chain comprising at least one,preferentially at least two, preferentially three, of CDR-H1, CDR-H2 andCDR-H3 of amino acid sequences SEQ ID No 4, 5 and 6, respectively, orsequences with at least 80%, preferably 85%, 90%, 95% and 98% identityafter optimal alignment with sequences SEQ ID No 4, 5 and 6,respectively, and a light chain comprising at least one, preferentiallyat least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 ofamino acid sequences SEQ ID No 7, 8 and 9, respectively, or sequenceswith at least 80%, preferably 85%, 90%, 95% and 98% identity afteroptimal alignment with sequences SEQ ID No 7, 8 and 9, respectively,

A humanized antibody comprising a heavy chain comprising at least one,preferentially at least two, preferentially three, of CDR-H1, CDR-H2 andCDR-H3 of amino acid sequences SEQ ID No 10, 11 and 12, respectively, orsequences with at least 80%, preferably 85%, 90%, 95% and 98% identityafter optimal alignment with sequences SEQ ID No 10, 11 and 12,respectively, and a light chain comprising at least one, preferentiallyat least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 ofamino acid sequences SEQ ID No 13, 14 and 15, respectively, or sequenceswith at least 80%, preferably 85%, 90%, 95% and 98% identity afteroptimal alignment with sequences SEQ ID No 13, 14 and 15, respectively,

A humanized antibody comprising a heavy chain comprising at least one,preferentially at least two, preferentially three, of CDR-H1, CDR-H2 andCDR-H3 of amino acid sequences SEQ ID No 16, 17 and 18, respectively, orsequences with at least 80%, preferably 85%, 90%, 95% and 98% identityafter optimal alignment with sequences SEQ ID No 16, 17 and 18,respectively, and a light chain comprising at least one, preferentiallyat least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 ofamino acid sequences SEQ ID No 19, 20 and 21, respectively, or sequenceswith at least 80%, preferably 85%, 90%, 95% and 98% identity afteroptimal alignment with sequences SEQ ID No 19, 20 and 21, respectively,

A humanized antibody comprising a heavy chain comprising at least one,preferentially at least two, preferentially three, of CDR-H1, CDR-H2 andCDR-H3 of amino acid sequences SEQ ID No 22, 23 and 24, respectively, orsequences with at least 80%, preferably 85%, 90%, 95% and 98% identityafter optimal alignment with sequences SEQ ID No 22, 23 and 24,respectively, and a light chain comprising at least one, preferentiallyat least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 ofamino acid sequences SEQ ID No 25, 26 and 27, respectively, or sequenceswith at least 80%, preferably 85%, 90%, 95% and 98% identity afteroptimal alignment with sequences SEQ ID No 25, 26 and 27, respectively,

A humanized antibody comprising a heavy chain comprising at least one,preferentially at least two, preferentially three, of CDR-H1, CDR-H2 andCDR-H3 of amino acid sequences SEQ ID No 28, 29 and 30, respectively, orsequences with at least 80%, preferably 85%, 90%, 95% and 98% identityafter optimal alignment with sequences SEQ ID No 28, 29 and 30,respectively, and a light chain comprising at least one, preferentiallyat least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 ofamino acid sequences SEQ ID No 31, 32 and 33, respectively, or sequenceswith at least 80%, preferably 85%, 90%, 95% and 98% identity afteroptimal alignment with sequences SEQ ID No 31, 32 and 33, respectively,and

A humanized antibody comprising a heavy chain comprising at least one,preferentially at least two, preferentially three, of CDR-H1, CDR-H2 andCDR-H3 of amino acid sequences SEQ ID No 34, 35 and 36, respectively, orsequences with at least 80%, preferably 85%, 90%, 95% and 98% identityafter optimal alignment with sequences SEQ ID No 34, 35 and 36,respectively, and a light chain comprising at least one, preferentiallyat least two, preferentially three, of CDR-L1, CDR-L2 and CDR-L3 ofamino acid sequences SEQ ID No 37, 38 and 39, respectively, or sequenceswith at least 80%, preferably 85%, 90%, 95% and 98% identity afteroptimal alignment with sequences SEQ ID No 37, 38 and 39, respectively,

wherein said antibody also comprises constant regions of the light-chainand the heavy-chain derived from a human antibody.

In another more particular embodiment, said antibody is a humanizedantibody selected in the group consisting of:

A humanized antibody comprising a heavy chain variable region of aminoacid sequence SEQ ID No 53, and a light chain variable region of aminoacid sequence SEQ ID No 54;

A humanized antibody comprising a heavy chain variable region of aminoacid sequence SEQ ID No 55, and a light chain variable region of aminoacid sequence SEQ ID No 56;

A humanized antibody comprising a heavy chain variable region of aminoacid sequence selected between SEQ ID No 57, 58, and 59, and a lightchain variable region of amino acid sequence selected between SEQ ID No60, 61, and 62;

A humanized antibody comprising a heavy chain variable region of aminoacid sequence selected between SEQ ID No 63, 64, and 65, and a lightchain variable region of amino acid sequence selected between SEQ ID No66, 67, and 68;

A humanized antibody comprising a heavy chain variable region of aminoacid sequence selected between SEQ ID No 69 and 71, and a light chainvariable region of amino acid sequence selected between SEQ ID No 70 and72; and

A humanized antibody comprising a heavy chain variable region of aminoacid sequence selected between SEQ ID No 75 and 76, and a light chainvariable region of amino acid sequence selected between SEQ ID No 77 and78;

wherein said antibody also comprises constant regions of the light-chainand the heavy-chain derived from a human antibody.

In a first embodiment, a method according to the invention comprisescontacting a biological sample with an anti-hPG antibody binding to anepitope of hPG, wherein said epitope is located within the C-terminalpart of hPG or to an epitope located within the N-terminal part of hPG.

In a more specific embodiment, a method according to the inventioncomprises contacting a biological sample with an anti-hPG antibodybinding to an epitope of hPG, wherein said epitope includes an aminoacid sequence corresponding to an amino acid sequence of the N-terminalpart of progastrin chosen among an amino acid sequence corresponding toamino acids 10 to 14 of hPG, amino acids 9 to 14 of hPG, amino acids 4to 10 of hPG, amino acids 2 to 10 of hPG and amino acids 2 to 14 of hPG,wherein the amino acid sequence of hPG is SEQ ID No 1.

In a more specific embodiment, a method according to the inventioncomprises contacting a biological sample with an anti-hPG antibodybinding to an epitope of hPG, wherein said epitope includes an aminoacid sequence corresponding to an amino acid sequence of the C-terminalpart of progastrin, chosen among an amino acid sequence corresponding toamino acids 71 to 74 of hPG, amino acids 69 to 73 of hPG, amino acids 71to 80 of hPG (SEQ ID No 40), amino acids 76 to 80 of hPG, and aminoacids 67 to 74 of hPG, wherein the amino acid sequence of hPG is SEQ IDNo 1.

In a first embodiment, a composition according to the inventioncomprises an antibody recognizing an epitope including an amino acidsequence corresponding to an amino acid sequence of progastrin.

In a more specific embodiment, a composition according to the inventioncomprises an antibody recognizing an epitope of progastrin wherein saidepitope includes an amino acid sequence corresponding to an amino acidsequence of the N-terminal part of progastrin, wherein said amino acidsequence may include residues 10 to 14 of hPG, residues 9 to 14 of hPG,residues 4 to 10 of hPG, residues 2 to 10 of hPG or residues 2 to 14 ofhPG, wherein the amino acid sequence of hPG is SEQ ID No 1.

In a more specific embodiment, a composition according to the inventioncomprises an antibody recognizing an epitope of progastrin wherein saidepitope includes an amino acid sequence corresponding to an amino acidsequence of the C-terminal part of progastrin, wherein said amino acidsequence may include residues 71 to 74 of hPG, residues 69 to 73 of hPG,residues 71 to 80 of hPG (SEQ ID No 40), residues 76 to 80 of hPG, orresidues 67 to 74 of hPG, wherein the amino acid sequence of hPG is SEQID No 1.

In a particular embodiment of a method for the in vitro diagnosis oflung cancer according to the invention, said method comprises a step ofcontacting a biological sample from a subject with a first moleculewhich binds to a first part of progastrin and with a second moleculewhich binds to a second part of progastrin. In a more particularembodiment, wherein said progastrin-binding molecule is an antibody, abiological sample from a subject is contacted with an antibody whichbinds to a first epitope of progastrin and with a second antibody whichbinds to a second epitope of progastrin.

In a particular embodiment of the method of the invention, said methodcomprises a step of contacting a biological sample from a subject with afirst agent which binds to a first part of progastrin and with a secondagent which binds to a second part of progastrin. In a more particularembodiment, wherein said progastrin-binding molecule is an antibody, abiological sample from a subject is contacted with an antibody whichbinds to a first epitope of progastrin and with a second antibody whichbinds to a second epitope of progastrin.

According to a preferred embodiment, said first antibody is bound to aninsoluble or partly soluble carrier. Binding of progastrin by said firstantibody results in capture of progastrin from said biological sample.Preferably, said first antibody is an antibody binding to an epitope ofhPG, wherein said epitope includes an amino acid sequence correspondingto an amino acid sequence of the C-terminal part of progastrin, asdescribed above. More preferably, said first antibody is monoclonalantibody Mab14, produced by hybridoma 2H9F4B7, described in WO2011/083088. Hybridoma 2H9F4B7 was deposited under the Budapest Treatyat the CNCM, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 ParisCEDEX 15, France, on 27 Dec. 2016, under reference 1-5158 (see WO2017/114973).

According to another preferred embodiment, said second antibody islabelled with a detectable moiety, as described below. Binding ofprogastrin by second antibody enables the detection of the progastrinmolecules which were present in the biological sample. Further, bindingof progastrin by second antibody enables the quantification of theprogastrin molecules which were present in the biological sample.Preferably, said second antibody is an antibody binding to an epitope ofhPG, wherein said epitope includes an amino acid sequence correspondingto an amino acid sequence of the N-terminal part of progastrin, asdescribed above. More preferably, said N-terminal antibody is apolyclonal antibody, as described above. Alternatively, it is alsopossible to use a monoclonal antibody biding an epitope within theN-terminus of progastrin, such as e.g. the N-terminus monoclonalantibodies described above, notably a monoclonal antibody comprising aheavy chain comprising CDR-H1, CDR-H2 and CDR-H3 of amino acid sequencesSEQ ID No 16, 17 and 18, respectively, and a light chain comprisingCDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No 19, 20 and21.

In a particularly preferred embodiment, the first antibody is bound toan insoluble or partly soluble carrier and the second antibody islabelled with a detectable moiety.

In a preferred embodiment, the method of the present invention for thediagnosis of lung cancer comprises the detection of progastrin in abiological sample from a human subject.

In a more preferred embodiment, the method of the present invention forthe diagnosis of lung cancer comprises the determination of theconcentration of progastrin in a biological sample from a human subject.

In another particular embodiment, the method of the present inventionfor the diagnosis of lung cancer comprises the detection of theconcentration of progastrin in a biological sample from a human subject,wherein said biological sample is selected from blood, serum and plasma.

In a further preferred embodiment, the method of the present inventioncomprises contacting a sample from said subject with an anti-hPGantibody as described above, wherein the binding of said anti-hPGantibody in the sample indicates the presence of lung cancer in saidsubject.

In a more particular embodiment, the method of the present inventioncomprises contacting a sample from said subject with an anti-hPGantibody as described above, wherein a concentration of progastrinsuperior to 10 pM in said plasma is indicative of the presence of lungcancer in said subject.

More preferably, the method of the present invention comprisescontacting a sample from said subject with an anti-hPG antibody asdescribed above, wherein a concentration of progastrin superior to 10pM, 20 pM, 30 pM or 40 pM in said sample is indicative of the presenceof lung cancer in said subject.

Still more preferably, the method of the present invention comprisescontacting a sample from said subject with an anti-hPG antibody asdescribed above, wherein a concentration of progastrin superior to 10pM, preferably to 20 pM, more preferably to 30 pM, still more preferablyto 40 pM, even more preferably to 50 pM in said sample is indicative ofthe presence of metastasized lung cancer in said subject

The present invention also relates to methods for monitoring theefficacy of a treatment for lung cancer in a patient, such aschemotherapy, biological therapy, immunotherapy or antibody therapy, bydetermining the concentration of progastrin in a first sample, such as abodily fluid or biopsy of lung cancer, obtained from a patient beforetreatment for lung cancer, and then comparing the concentration ofprogastrin in the first sample to that in a second sample obtained fromthe same patient after treatment, where a reduction in the concentrationof progastrin in said second sample compared to said first sampleindicates that the treatment was effective.

In a particular embodiment, a method according to the inventioncomprises comparing the concentration of progastrin in a biologicalsample obtained from a patient with a predetermined value ofconcentration of progastrin in the sample, in a more particularembodiment, said predetermined value is chosen among: an mean, oraverage, of sample values based on the mean, or average, determinationof the value in a population free of lung cancer, a progastrinconcentration value obtained when the patient was known to be free oflung cancer.

In a particular embodiment, a method according to the invention for thein vitro diagnosis of lung cancer comprises the determination ofprogastrin concentration in a sample from said patient and a seconddiagnosis test of lung cancer. In a more particular embodiment, a methodaccording to the invention for the in vitro diagnosis of lung cancercomprises the determination of progastrin concentration in a sample fromsaid patient and a second diagnosis test of lung cancer, wherein saidsecond diagnosis test comprises the detection of a particular biomarkerchosen among: carcinoembryonic antigen (CEA), neuron-specific enolase(NSE), cytokeratin 19 (CYFRA-21-1), alpha-fetoprotein, carbohydrateantigen-125 (CA-125), carbohydrate antigen-19.9 (CA-19.9), and ferritin,independently or in combination (Li et al, 2012).

In a particular embodiment of the invention, a method according to thepresent invention comprises the determination of the level of progastrinover time in samples from a patient who has been or is being treated forlung cancer.

The characteristics of the embodiments of the invention will becomefurther apparent from the following detailed description of examplesbelow.

FIGURE LEGENDS

FIG. 1

Progastrin concentration was measured in 40 plasma samples from lungcancer patients and 119 plasma samples from healthy donor using theELISA Kit DECODE Lab (capture antibody: Mab14, detection antibody:anti-hPG polyclonal).

EXAMPLES Example 1: Detection of Plasmatic Progastrin ConcentrationUsing Polyclonal Antibodies

Plasma progastrin levels were quantified by ELISA through the use of twospecific anti-progastrin antibodies: capture antibodies are coated onthe wells of the plate, whereas revelation antibodies are used to detectprogastrin and mediates revelation of the signal.

In the present example, quantification is based on the ELISA methodwhich allows, through the use of a substrate whose reaction emits light,to assign a value proportional to the luminescence amount of antibodiesbound to the antigen retained by capture antibodies.

Material

Reagents and apparatus are listed in Table 7:

TABLE 7 Désignation Provider Référence Plates MaxiSORP white Nunc, 96wells Dutscher # 055221 Sodium Carbonate/Bicarbonate Sigma # 21851 DPBS1× Lonza # P04-36500 Tween-20 Biosolve # 20452335 BSA Euromedex #04-100-810-C Streptavidin-HRP Pierce # 21130 (Thermo) SuperSignal ELISAFemto Maximum Pierce # 37074 Sensitivity Substrate (Thermo)Anti-ProGastrin Polyclonal Antibody Eurogentec /

Polyclonal antibodies were obtained by immunizing a rabbit withN-terminal progastrin (SEQ ID No 2) or with C-terminal progastrincorresponding to amino acids 71 to 80 of hPG and having the sequenceFGRRSAEDEN (SEQ ID No 40), according to standard protocols.

The binding characteristics of polyclonal antibodies against progastrinused in this assay are the following: absence of binding to G34-Gly,G34, G17-Gly, G17, binding to full length progastrin.

96 wells plates are coated by preparing a solution of carbonate-sodiumbicarbonate, 50 mM pH 9.6 by dissolving the contents of one capsule in100 ml of MilliQ water. A solution of capture antibody (3 μg/ml),corresponding to polyclonal antibodies obtained by using the C-terminalof progastrin FGRRSAEDEN (SEQ ID No 40) is prepared in carbonate buffer.100 microliters of antibodies solution is added to each well andincubated at 4° C. for 16 hours (1 night). Plates are then blocked byeliminating the antibodies solution and wash 3 times with 300 μl1×PBS/0.1% Tween-20, then adding 200 μl of blocking buffer (1×PBS/0.1%Tween-20/0.1% BSA) per well, and incubated 2 hours at 22° C. Blockingbuffer is then eliminated, wells are washed 3 times with 300 μl1×PBS/0.1% Tween-20.

Plasma dilution is performed as follows: The plasma is used pure,diluted ½, ⅕ and 1/10. Dilutions are prepared from pure plasma in1×PBS/0.1% Tween 20/0.1% BSA.

For the control test, ELISA in the presence of a known concentration ofprogastrin, progastrin dilution is prepared as follows: stockrecombinant PG (Full length human progastrin produced in E. coli andaffinity purified with Glutathione agarose/Tag removal (Tev)/IMACCounter purification/dialysis, from Institut Pasteur, Paris, France) isprepared at a concentration of 0.45 mg/ml (45 microM), in triplicate.Ranges of progastrin concentrations were prepared as follows:

-   -   Solution A: Pre-dilution 1/10, 2 μl of stock+18 μl of the buffer    -   Solution B: Pre-dilution 1/100, 10 μl of A+90 μl of the buffer    -   Solution C: Pre-dilution 1/1000, 10 μl of B+90 μl of the buffer    -   Solution D: 500 pM, 5.55 μl of C+494.5 μl of the diluent    -   Solution E: 250 pM, 250 μl of D+250 μl of the diluent    -   Solution F: 100 pM, 200 μl of E+300 μl of the diluent    -   Solution G: 50 pM, 250 μl of F+250 μl of the diluent    -   Solution H: 25 pM, 200 μl of G+200 μl of the diluent    -   Solution I: 10 pM, 100 μl of H+150 μl of the diluent

The range of recombinant PG is linear and can therefore be more or lessextensive according to the antibody used.

For the preparation of test samples, approximately 500 μl of each sampleare set aside and stored until analysis (and confirmation if necessary)of the results. 100 μl of each point of the range and/or plasmas areassayed pure, diluted to ½, ⅕ and 1/10, and incubated for 2 hours at 22°C. on the plates.

For the revelation of the test, the plates are washed 3 times with 300μl 1×PBS/0.1% Tween-20. A solution of the polyclonal rabbitanti-progastrin antibody, wherein said antibodies have been obtained byusing the N-terminal part of progastrin as an immunogen, coupled tobiotin to 0.5 μg/ml, is prepared by dilution in 1×PBS/0.1% Tween-20/0.1%BSA. 100 μl of this solution is added to each well. Incubation takesplace for 1 hour at 22° C. The revelation with streptavidin-HRP isperformed by removing detection antibody and wash 3 times with 300 μl1×PBS/0.1% Tween-20, then preparing a solution of Streptavidin-HRP at 20ng/ml diluted in 1×PBS/0.1% Tween-20/0.1% BSA, wherein 100 Add 100 μl ofthis solution is added to each well, before incubation for 1 hour at 22°C.

The detection consists of eliminating streptavidin-HRP and wash 3 timeswith 300 μl 1×PBS/0.1% Tween-20, then adding 100 μl of chemiluminescentsubstrate solution per well. The substrate solution is prepared bymixing equal volumes of the two solutions SuperSignal ELISA Femto kit,20 ml+20 ml, 30 minutes before use and stored at room temperature in thedark. Luminescence is read after 5 minutes incubation at roomtemperature in the dark.

For each condition, the test is performed in triplicate and the resultsof the ranges will be presented as a graph showing the change inluminescence depending on the progastrin concentration. For each plasmadilution, the concentration of progastrin is determined using theequation of the linear regression line of the corresponding range (range1/10th for a sample diluted to 1/10th).

Methods and Results

The median plasmatic concentration of progastrin is 0 pM in controlpatients (n=103), whereas a significant plasmatic concentration ofprogastrin can be detected in patients having lung cancer. Thus,patients with lung cancer have higher levels of progastrin in theirplasma compared to healthy control individuals.

Example 2: Detection of Progastrin Concentration Using MonoclonalAnti-Progastrin Antibodies

The wells of Nunc MaxiSORP 96-well plates are coated with a firstprogastrin-specific antibody as follows. Anti-progastrin monoclonalantibodies specific for the carboxy-terminal region of progastrin arediluted to a concentration of 3 μg/ml in a solution of 50 mM, pH 9.6sodium carbonate/bicarbonate buffer in MilliQ water.

A total of 100 μl of the antibody solution is then added to each well ofthe 96-well plates, and incubated overnight at 4° C. After binding, theantibody solution is removed from the wells, which are then washed threetimes with 100 μl wash buffer (1×PBS/0.1% Tween-20). A total of 100 μlblocking buffer (1×PBS/0.1% Tween-20/0.1% BSA) is then added to eachwell and incubated for 2 hours at 22° C. Blocking buffer is then removedand the wells washed three times with wash buffer. Plasma or serumsamples isolated from patients is then added to the wells in a volume of100 μl in a dilution series, typically 1:1, 1:2, 1:5 and 1:10 dilutions,and is then incubated for 2 hours at 22° C. Plasma or serum samples areanalyzed in duplicate.

Assays also include two standard curves. The first standard curve isprepared using dilutions of recombinant progastrin to a final amount of1 ng, 0.5 ng, 0.25 ng, 0.1 ng, 0.05 ng, 0.01 ng, and 0 ng per well. Thesecond standard curve, which serves as a negative control, is preparedfrom progastrin-negative human serum diluted in blocking buffer at thesame dilutions as the test samples, i.e., 1:1, 1:2, 1:5 and 1:10.Alternatively, when plasma samples are being assayed, the secondstandard curve, which serves as a negative control, is prepared fromprogastrin-negative human plasma diluted in blocking buffer at the samedilutions as the test samples, i.e., 1:1, 1:2, 1:5 and 1:10.

After incubation with the plasma or serum samples is complete, the wellcontents are removed and the wells are washed three times with washbuffer, 100 μl/well, after which progastrin bound to the first antibodyis detected using a second antibody specific for progastrin, as follows.

Biotin-coupled anti-progastrin monoclonal antibodies specific for theamino-terminal region of progastrin are diluted in blocking buffer to aconcentration of 0.1 to 10 μl g/ml, depending on the antibody. A totalof 100 μl of the antibody solution is then added to each well, andincubated for 1 hour at 22° C.

After secondary antibody binding is complete, the plates are washedthree times with wash buffer, 100 μl/well, after which 100 μl of asolution of streptavidin-HRP (25 ng/ml in blocking buffer) is added toeach well and incubated for 1 hour at 22° C. After incubation with thestreptavidin-HRP solution is complete, the plates are washed three timeswith wash buffer, 100 μl/well. Thereafter, 100 μl of chemiluminescentsubstrate prepared using a Pierce SuperSignal ELISA Femto MaximumSensitivity Chemiluminescent Substrate kit, is added per well, incubatedfor 5 min at room temperature in the dark, and then read on aluminometer.

Based on the luminometer readings, linear regression analysis is used toderive the equation of the lines corresponding to the standard curvedata. Using this equation, the concentration of progastrin in thevarious patient samples is then calculated.

The median plasmatic concentration of progastrin is calculated inpatients having lung cancer and compared to the median plasmaticconcentration of progastrin in plasma of control patients. Patients withlung cancer had elevated levels of progastrin in their plasma comparedto healthy control individuals.

Example 3: Detection of Plasmatic Progastrin Concentration Using aCombination of Polyclonal Antibodies and Monoclonal Antibodies

In the present example, plasma progastrin levels are quantified by ELISAthrough the use of antibody specific for human progastrin (hPG)pre-coated on a 96-well plate. Standards and samples are added to thewells, and any hPG present binds to the immobilized capture antibody.The wells are washed and an anti-hPG detection antibody horseradishperoxidase (HRP) conjugate is added, producing anantibody-antigen-antibody “sandwich.” After a second wash, TMB substratesolution is added, which produces a blue color in direct proportion tothe amount of hPG present in the initial sample. The Stop Solutionchanges color from blue to yellow, and the wells are read at 450 nm witha microplate reader.

Polyclonal antibodies are obtained by immunizing a rabbit withN-terminal progastrin (SEQ ID No 2) or with C-terminal progastrincorresponding to amino acids 71 to 80 of hPG and having the sequenceFGRRSAEDEN (SEQ ID No 40), according to standard protocols.

Monoclonal antibodies are obtained by using hybridomas producingantibodies against N-terminal progastrin (SEQ ID No 2) or againstC-terminal progastrin corresponding to amino acids 71 to 80 of hPG andhaving the sequence FGRRSAEDEN (SEQ ID No 40), according to standardprotocols.

The binding characteristics of polyclonal and monoclonal antibodiesagainst progastrin used in this assay are the following: absence ofbinding to G34-Gly, G34, G17-Gly, G17, binding to full lengthprogastrin.

For the control test, ELISA in the presence of a known concentration ofprogastrin, progastrin dilution is prepared as follows: stockrecombinant PG (Full length human progastrin produced in E. coli andaffinity purified with Glutathione agarose/Tag removal (Tev)/IMACCounter purification/dialysis, from Institut Pasteur, Paris, France) isprepared at a concentration of 0.45 mg/ml (45 microM), in triplicate.Ranges of progastrin concentrations are prepared as follows:

Solution A: Pre-dilution 1/10, 2 μl of stock+18 μl of the buffer

Solution B: Pre-dilution 1/100, 10 μl of A+90 μl of the buffer

Solution C: Pre-dilution 1/1000, 10 μl of B+90 μl of the buffer

Solution D: 500 pM, 5,55 μl of C+494.5 μl of the diluent

Solution E: 250 pM, 250 μl of D+250 μl of the diluent

Solution F: 100 pM, 200 μl of E+300 μl of the diluent

Solution G: 50 pM, 250 μl of F+250 μl of the diluent

Solution H: 25 pM, 200 μl of G+200 μl of the diluent

Solution I: 10 pM, 100 μl of H+150 μl of the diluent

The range of recombinant PG is linear and can therefore be more or lessextensive according to the antibody used.

Methods and Results

Progastrin levels are determined in plasma samples from subjects whowere known to have developed lung cancer later. Progastrin is capturedwith the C-terminus monoclonal antibody mAb 14 produced by hybridoma2H9F4B7 described in WO 2011/083088 (Hybridoma 2H9F4B7 is depositedunder the Budapest Treaty at the CNCM, Institut Pasteur, 25-28 rue duDocteur Roux, 75724 Paris CEDEX 15, France, on 27 Dec. 2016, underreference 1-5158.). Detection is performed with labelled polyclonalantibodies specific for the N-terminus.

The control is constituted by plasma samples from the generalpopulation.

The data demonstrate that patients with lung cancer have detectablelevels of progastrin in their plasma whereas healthy control individualshave none.

Example 4: Detection of Plasmatic Progastrin Concentration Using DECODELab Kit

The test allows a measurement of hPG in plasma EDTA by ELISA.

The kit utilizes a capture antibody specific for hPG pre-coated on a96-well plate. hPG present in standards and samples added to the wellsbind to the immobilized capture antibody. The wells are washed and ananti-hPG detection antibody horseradish peroxidase (HRP) conjugate isadded, resulting in an antibody-antigen-antibody complex. After a secondwash, a 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solution is addedto the well, producing a blue color in direct proportion to the amountof hPG present in the initial sample. The Stop Solution changes thecolour from blue to yellow, and the wells are read at 450 nm with amicroplate reader.

Methods and Results

40 plasma samples from lung cancer patients and 119 plasma samples fromhealthy donor were used to measure the concentration of progastrin usingthe ELISA Kit DECODE Lab (capture antibody: Mab14, detection antibody:anti-hPG polyclonal) following manufacturer's recommendation.

Briefly:

-   -   1. Prepare all reagents, controls, and samples as directed in        the previous section except the 1× Conjugate.    -   2. Remove excess strip from the microtiter plate frame, return        them to the plate packet and store at 2-8° C.    -   3. Samples and controls must be tested in duplicate. Prepare the        pre-loading of controls and samples by adding 65 μl/replicate in        wells of the 96-Well DeepWell Polypropylene Microplates.    -   4. Add 50 μl of Sample dilution buffer to all the wells that        will be used from the 96 pre-coated well plate strips included        in the kit.    -   5. Transfer 50 μl of the controls and samples with a        multi-channel pipette (8 channels) from the pre-loading 96-Well        DeepWell Polypropylene Microplates to the 96 pre-coated well        plate strips included in the kit. The loading time should not        exceed 10 minutes.    -   6. Cover the plate with plastic paraffin and incubate for 3 h±5        min at 37° C. (±2° C.).    -   7. Prepare the 1× Conjugate as described in section 10.2    -   8. At the end of the incubation step, discard all the liquid        from the wells by inverting the plate. Proceed to a thorough        washing step by adding 300 μl per well of 1× Wash solution.        Discard the 1× wash solution by inverting the plate and        thoroughly pat dry the microtiter plate frame upside down on        absorbent paper. Repeat the washing step 6 times. At the end of        the washing steps, ensure the complete removal of the liquid        from the wells: all liquid has been successfully removed when no        sign of liquid remains on the paper towel. The wash procedure is        critical. Insufficient washing may result in poor precision and        falsely elevated absorbance readings.    -   9. Add 100 μl of the 1× Conjugate to each well.    -   10. Cover the plate with plastic paraffin and incubate 30 min±3        min at 21° C. (±5° C.).    -   11. At the end of the incubation step, discard all the liquid        from the wells by inverting the plate. Proceed to a thorough        washing step by adding 300 μl per well of 1× Wash solution.        Discard the 1× wash solution by inverting the plate and        thoroughly pat dry the microtiter plate frame upside down on        absorbent paper. Repeat the washing step 6 times. At the end of        the washing steps, ensure the complete removal of the liquid        from the wells: all liquid has been successfully removed when no        sign of liquid remains on the paper towel. The wash procedure is        critical. Insufficient washing will result in poor precision and        falsely elevated absorbance readings.    -   12. Add 100 μl of the Substrate solution to each well. Upon the        addition of the Substrate solution, the content of the Positive        Control 1 and Positive Control 2 wells should become blue.    -   13. Incubate for 15 min±2 min at 21° C. (±5° C.) in the dark.    -   14. Without removing the content, of the wells, add 100 μl of        the Stop solution to each well in order to stop the reaction.        Upon the addition of the Stop solution, the content of the        Positive Control 1 and Positive Control 2 wells should become        yellow.    -   15. Read and record the O.D. at 450 nm.

As shown in FIG. 1, the median plasmatic concentration of progastrin was0 pM in control patients (n=119), whereas a significant plasmaticconcentration of progastrin could be detected in patients having lungcancer (n=40). Thus, patients with lung cancer have higher levels ofprogastrin in their plasma compared to healthy control individuals.

BIBLIOGRAPHIC REFERENCES

Yanaoka et al, Cancer Epidemiol Biomarkers Prey, 2008, 17(4)

Pepe et al, J Natl Cancer Inst, 2008, October, 100(20)

-   -   Leja et al, Best Practice a Research Clinical Gastroenterology,        2014, Dec. 28(6)

1. A method for the in vitro diagnosis of lung cancer in a subject,comprising the steps of: a) contacting said biological sample from saidsubject with at least one progastrin-binding molecule, b) detecting thebinding of said progastrin-binding molecule to progastrin in saidsample, wherein said binding indicates the presence of lung cancer insaid subject.
 2. The method of claim 1, wherein step b) furthercomprises determining the concentration of progastrin and wherein aconcentration of progastrin at least 10 pM in said biological sample isindicative of the presence of lung cancer in said subject.
 3. The methodof claim 2, comprising the further steps of: c) determining a referenceconcentration of progastrin in a reference sample, d) comparing theconcentration of progastrin in said biological sample with saidreference concentration of progastrin, e) determining, from thecomparison of step d), the presence of lung cancer.
 4. The method of anyone of claims 1 to 3, wherein said progastrin-binding molecule is anantibody, or an antigen-binding fragment thereof.
 5. The method of anyof claims 1 to 4, wherein said antibody, or antigen-binding fragmentthereof, is selected among N-terminal anti-progastrin monoclonalantibodies and C-terminal anti-progastrin monoclonal antibodies.
 6. Themethod of any of claims 1 to 5, wherein said antibody binding toprogastrin is a monoclonal antibody chosen in the group consisting of: Amonoclonal antibody comprising a heavy chain comprising at least one,preferentially at least two, preferentially three, of CDR-H1, CDR-H2 andCDR-H3 of amino acid sequences SEQ ID No 4, 5 and 6, respectively, and alight chain comprising at least one, preferentially at least two,preferentially three, of CDR-L1, CDR-L2 and CDR-L3 of amino acidsequences SEQ ID No 7, 8 and 9, respectively, A monoclonal antibodycomprising a heavy chain comprising at least one, preferentially atleast two, preferentially three, of CDR-H1, CDR-H2 and CDR-H3 of aminoacid sequences SEQ ID No 10, 11 and 12, respectively, and a light chaincomprising at least one, preferentially at least two, preferentiallythree, of CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No13, 14 and 15, respectively, A monoclonal antibody comprising a heavychain comprising at least one, preferentially at least two,preferentially three, of CDR-H1, CDR-H2 and CDR-H3 of amino acidsequences SEQ ID No 16, 17 and 18, respectively, and a light chaincomprising at least one, preferentially at least two, preferentiallythree, of CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No19, 20 and 21, respectively, A monoclonal antibody comprising a heavychain comprising at least one, preferentially at least two,preferentially three, of CDR-H1, CDR-H2 and CDR-H3 of amino acidsequences SEQ ID No 22, 23 and 24, respectively, and a light chaincomprising at least one, preferentially at least two, preferentiallythree, of CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No25, 26 and 27, respectively, A monoclonal antibody comprising a heavychain comprising at least one, preferentially at least two,preferentially three, of CDR-H1, CDR-H2 and CDR-H3 of amino acidsequences SEQ ID No 28, 29 and 30, respectively, and a light chaincomprising at least one, preferentially at least two, preferentiallythree, of CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No31, 32 and 33, respectively, A monoclonal antibody comprising a heavychain comprising at least one, preferentially at least two,preferentially three, of CDR-H1, CDR-H2 and CDR-H3 of amino acidsequences SEQ ID No 34, 35 and 36, respectively, and a light chaincomprising at least one, preferentially at least two, preferentiallythree, of CDR-L1, CDR-L2 and CDR-L3 of amino acid sequences SEQ ID No37, 38 and 39, respectively, and A monoclonal antibody produced by thehybridoma deposited at the CNCM, Institut Pasteur, 25-28 rue du DocteurRoux, 75724 Paris CEDEX 15, France, on 27 Dec. 2016, under reference1-5158.
 7. The method of any one of claims 1 to 6, wherein thedetermination of step a) includes: (i) contacting said sample with afirst progastrin-binding molecule which binds to a first part ofprogastrin, and (ii) contacting said sample with a secondprogastrin-binding molecule which binds to a second part of progastrin.8. The method of claim 7, wherein the first progastrin-binding moleculebinds an epitope within the C-terminus of progastrin.
 9. The method ofany one of claim 7 or 8, wherein said progastrin-binding molecule is amonoclonal antibody produced by the hybridoma deposited at the CNCM,Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris CEDEX 15,France, on 27 Dec. 2016, under reference 1-5158.
 10. The method of anyone of claims 7 to 9, wherein the second progastrin-binding moleculebinds an epitope within the N-terminus of progastrin.
 11. The method ofany one of claims 7 to 10, wherein said second progastrin-bindingmolecule is a polyclonal antibody binding an epitope within theN-terminus of progastrin or a monoclonal antibody comprising a heavychain comprising the following three CDRs, CDR-H1, CDR-H2 and CDR-H3 ofamino acid sequences SEQ ID No 16, 17 and 18, respectively, and a lightchain comprising the following three CDRs, CDR-L1, CDR-L2 and CDR-L3 ofamino acid sequences SEQ ID No 19, 20 and 21, respectively.
 12. Themethod of any one of claims 1 to 11, wherein the level of progastrin isdetermined in step a) with an ELISA.
 13. The method of any one of claims1 to 6, wherein said biological sample is contacted with a firstmolecule, which binds to a first part of progastrin, and with a secondmolecule, which binds to a second part of progastrin.
 14. The method ofany one of claims 1 to 7, wherein said biological sample is chosenamong: blood, serum and plasma.
 15. The method of any one of claims 1 to8, wherein said biological sample is plasma, and wherein a concentrationof progastrin of at least 10 pM is indicative of the presence of lungcancer in said subject.
 16. Use of a progastrin-binding antibody, or anantigen-binding fragment thereof, as in any one of claims 10 to 14 forthe in vitro diagnosis of lung cancer.